Neural stem cells have a fundamental role in generating cellular diversity in the developing mammalian nervous system. However, there is very little known about how neural stem cells are formed initially in embryogenesis. Evidence from studies primarily in Xenopus suggest that the acquisition of a neural fate in ectoderm cells is actively repressed and that escaping the repressive signal is the predominant mechanism by which cells reveal their default neural identity (Hemmati-Brivanlou and Melton, 1997). However, it is uncertain whether default neural specification occurs in mammalian development, and if so whether the process of default neural fate specification is homologous among vertebrate species.
During mouse gastrulation cells derived from the embryonic ectoderm are organized into either neural or epidermal primordia. The concept of vertebrate neural induction, borne out of studies in amphibian embryology, was proposed to account for the segregation of these two vertebrate ectodermal lineages (Spemann and Mangold, 1924; Waddington and Schnidt, 1933; Oppenheimer, 1936; Beddington, 1994). It was postulated that the nascont embryonic ectoderm received a positive inducing signal from the dorsal organizer tissue during gastrulation, which caused the ectodermal cells to adopt a neural fate in a restricted manner. In the absence of this signal, ectodermal cells were presumed to differentiate into epidermis, independent of any cellular communication.
Results from in vitro experiments of isolated ectodermal (animal cap) cells derived from amphibian gastrula supported a different model for neural fate specification. Prolonged low-density dissociation of ectodermal cells, in the absence of organizer tissue, resulted in most of the cells expressing neural marker or forming neural structures upon reaggregation (Godsave and Slack, 1989; Grunz and Tacke, 1989; Sato and Sargent, 1989). Furthermore, ectodermal explants (undissociated cells (expressing a dominant-negative receptor for activin (a member of the TGFβ superfamily of growth factors) were shown to become neural when cultured in vitro (Hemmati-Brivanlou and Melton, 1994). In studies aimed at identifying the nature of the organizer signals, molecules isolated from mesendodermal tissue, such as noggin and chordin, were found to be sufficient for inducing a second neural axis in analogous ectopic experiments performed in Xenopus (Smith et al., 1993; Sasai et al., 1995). However, the biochemical mechanism by which organizer signals promoted neural differentiation of ectodermal cells was not entirely consistent with a positive induction model for neural fate determination. Noggin and chorein were shown to act by binding extracellularly to bone morphogenetic proteins (BMPs), members of the TGFβ superfamily of molecules that strongly inhibit neural differentiation (Hemmati-Brivanlou and Melton, 1994). Thus, in a restricted manner, noggin and chordin prevent the binding of BMPs to their cognate receptors expressed on the surface of ectodermal cells (Piccolo et al., 1996; Zimmerman et al., 1996). In fact, BMP4 was shown to act as a positive signal for epidermal fate determination in the Xenopus ectoderm (Wilson and Hemmati-Brivanlou, 1995). These findings from amphibian experiments were consistent with the notion that the establishment of neural identity from the uncommitted ectoderm occurs by default (i.e. a state achieved autonomously after the removal of the inhibitory signals) in the absence of neural-inducing factors emanating from the organizer.
Embryonic stem (ES) cells are precursors to all embryonic lineages. ES cells are derived from the inner cell mass (ICM) of the pre-implanation mouse embryo (Evans and Kaufman, 1981; Martin, 1981) and can be sustained in an undifferentiated state in vitro while maintaining ICM characteristics. In prior studies of the neuronal differentiation of embryonic stem (ES) cells each experiment was preceded by embryoid body (EB) formation in the presence of serum. As a result, the derivation of neural cells was accomplished indirectly and under conditions where many culture media parameters are unknown.
It is desirable to have a method for differentiating ES cells toward neural cells more directly. Furthermore, it is desirable to have a model system with known constituents and well defined end products for the differentiation of neural cells from ES cells. Such as system would be useful in analyzing the role of single genes in the regulation of neural development, and for the development and testing of drugs for the treatment of developmental and cerebral neural anomalies and neuropathies.